While the discovery of antibiotics has revolutionized medicine, the continuous evolution of antibiotic- resistant infections threatens to reverse the advances made in treating infectious disease. Thus, the investigation of new drugs for the treatment of resistant infections is of paramount importance. Bacteria typically acquire resistance to 2-lactam antibiotics by producing 2-lactamases, enzymes that chemically inactivate the drug. The carbapenem class of antibiotics are much more resistant to 2-lactamases than the penicillins and cephalosporins, and as such they are clinically valuable in the treatment of resistant infections. This proposal outlines a strategy for studying the reaction mechanism of Carbapenem Synthase (CarC). This enzyme is involved in the biosynthesis of (5R)-carbapenem-3-carboxylic acid, which forms bicyclic nucleus of all carbapenem molecules. While carbapenems are produced naturally, there are no fermentative or semisynthetic methods for the production of carbapenem drugs, which are instead prepared by costly total synthesis. This is due in large part to the difficulties encountered in elucidating the biosynthetic pathway of thienamycin, the most potent and medicinally relevant natural carbapenem. The development of a biosynthetic or semisynthetic method for producing carbapenem compounds would increase the availability of existing drugs, in addition to facilitating the exploration of semisynthetic analogs in pursuit of novel therapies. Engineering CarC to accept non-natural substrates is an attractive approach to developing a chemoenzymatic synthesis of carbapenem drugs. A detailed mechanistic understanding of CarC will inform efforts to engineer synthetically useful enzyme variants. These studies are particularly important since structural studies on the CarC enzyme leave many questions unanswered about the precise nature of interactions between the substrate and active site. .